Pellet for administering gonadotropic pituitary hormones



Patented Dec. 31, 1946 PELLET FOR ADMINISTERING GONADO- TROPIC PITUITARY HORMONES Francis J. Saunders, Skokie, and Albert L. Raymond, Northfield, Ill., assignors to G. D. Searle & 00., Skokie, 111., a corporation of Illinois No'Drawing. Application February 27,1943, Serial No. 477,454

6 Claims. 1

This invention relates to a method of administering the g'onadotropic fractions of the her mones of the anterior pituitary gland, more particularly to a method which causes a very slow constant absorption of the active hormone ma terial by the tissues, and to a solid, relatively nonabsorbable preparation of these fractions, suitable for subcutaneous implantation, for use in this method of administration. The present application is a continuation-in-part of our. copending application, Serial No. 391,456, filed May 2, 1941, and of an earlier application, Serial No. 361,950, filed October 19, 1940, of which the firstmentioned application was a continuation-inpart.

It is well known thatmost ductless glands function in a substantially continuous manner, secret-- ving their hormones into the blood gradually, as

needed by, the body. The rate of secretion may vary widelywith circumstances, but in no case does a process of natural. secretion even remotely resemble the common method or therapeutic administration which is to present relatively massive doses of the hormone, in relatively readily absorbable form, and at relatively distant intervals. Such a method of administration results in the temporary presence of excessive amounts of hormone, followedby longer periods when the hormone is present in insufficient amount or may be entirely absent. It is one object of this invention to provide a means of administration of the anterior pituitary gonadotropichormones which resembles natural secretion in that there is a slow, more or' less uniform and prolonged release of the active material, from depots which may be established at relatively infrequent intervals. This leads to the further practical result that clinical use of these hormone fractions is simplified and rendered more satisfactory by requiring less frequent treatment at the hands of the practitioner. 7

It is recognized, further, that the common method of administration of the anterior pituitary gonadotropic hormone, as referred to above; is wasteful of hormone substance in that relatively large doses must ordinarily be administered in order to prolong the effect for more than a very short interval, much of the excess being quickly destroyed due to the labile nature of this hormone substance. It is another object of this invention'to provide a method of administration of this hormonewhich requires, in many instances, distinctly smaller doses than methods heretofore in use to achieve the desired effects.

'Closely related to'this object is a: further one,

' hundred times the amount required to produce the end point when administered according to the assay directions, have been totally ineffective in producing this end point when given in asingle dose. Thus, it is seen that response to a given amount of this hormone is increased by administration of the total dose of hormone in an increasing number of injections each of decreasing size... .A. further object of this invention is to present this hormone in a form which is maximally effective, that is; where the total dose may be considered-as being divided into an infinite number of infinitesimaldoses.

Another closely allied object of this invention is to increase the maximum response obtainable in'the use ofthese hormone fractions, regardless of dose. It is a widely recognized principle in pharmacology that, while increasing doses of a medicament may elicitmore or less proportionate increases in response from the organism being treated, yet this proportionation ordinarily holds only over a limited range of doses. When the upper 'limitof this range-has been exceeded, the increasein response becomes less and less, until finally there is obtained a maximal'response, which cannot be increased by any dosage. This phenomenon of maximal response is observed in ..-the "administration of the anterior pituitary gonadotropic hormones in experimental animals.

However, by. the use of the invention described herein, we. have beenyable consistently to produce responses in experimental animals: exceeding the maximum which we have heretofore been able-to obtain by standard injection techniques.

A further object of this invention isto provide a practical. convenientmethod of marketing these hormone fractionsin a completely stable condition. Itis acknowledged that aquemones is by injection of the aqueous solution, a manufacturer desiring to offer a stable product must prepare and market a dry powder in ampoules suitable for the extemporaneous preparation by the physician of a solution for injection. This is frequently a diflicult and expensive pro cedure for the manufacturer, and the preparation of the solution is ordinarily a source of annoyance to the physician. By the use of the invention herein described, these objections are largely overcome and a convenient stable product is made available.

We have discovered that all of the above objects, as well as others which may become apparent elsewhere in this disclosure, may be attained by the administration of the gonadotropic gland in the form of a pellet, which pellet consists of a dispersion of the desiccated, powdered, watersoluble hormone substance in a waxy me: dium which is solid at body temperature and relatively non-absorbable by body tissues. This pellet is implanted under the skin of the individual under treatment, resulting, in spite of the relative non-absorbability of the medium in which the hormone is dispersed, in a striking manifestation of the hormone effectaas can be quantitatively demonstrated in experimental animals. This manifestation of eifect is striking, unusual,

and unexpected, not only in its duration (for it is commonly accepted that individual doses of anterior pituitary gonadotropic hormones are comparatively evanescent and must be frequently repeated to become effective), but also in its magnitude of response, as will be shown in the table cited below. 7

We have found that the nature of this waxy dispersing medium exerts considerable influence upon the results which may be obtained by this method of administration. It is important that the medium be not fluid at body temperature, for we have found that fluid media permit too ready absorption of the hormone substance, thereby preventing the attainment of the abovestated objects of this invention.

The terms wax and waxy as used in this specificationand in the appended claims are not to be construed as referring only to substances rubbers may possess some of the properties enumerated above, they are not to be considered as coming within the scope of this definition. The following substances are illustrative of the waxes contemplated as dispersion media in the practice of this invention, though the invention is not to be construed as limited thereto: beeswax, bayberry wax, Chinese wax, paraflin wax, ceresin, stearin, palmitin, tallow, myristin, spermaceti, Japan wax, cetyl alcohol, stearic acid, propylene glycol monostearate, glycerol monostearate, oleostearin, hydrogenated coconut oil, and a variety of synthetic substitutes now offered on the market and in use at the present time. Among the latter may be mentioned the waxes currently ,7 sold under the following trade names: Gellofraction of the hormones of the anterior pituitary of the chemical class ordinarily defined as the esters of long-chain alcohols with long-chain fatty acids, but it is intended to include substances of similar physical properties of whatever chemical composition, such as the hard fats, paraflin (and other mineral waxes derived from pctroleum) and the so-called synthetic waxes of various chemical types. The waxy substances which we have found useful as dispersion media may be recognized by these common properties: they are insoluble in water (though generally soluble in such organic solvents as ether and chloroform); they soften appreciably before melting when warmed; they are, in general, greasy to the touch; they tend to spread or flow rather than disintegrate during such an operation as grinding; their. melting point lies above 40 centigrade;

they are substantially amorphous in nature. With respect to the latter limitation, it isrecognized that certain of the waxes (particularly the synthetic waxes) contemplated within this inwax, Flexowax C, Glycowax A, Nipocer,

BZ Wax A, Ceraflux. For illustration of the chemical composition of these waxes, Flexowax C is a hydrocarbon wax made up of approximately per cent by weight of carbon and approximately 14 per cent by weight of hydrogen, while BZ Wax A is a modified hydrocarbon wax made up of approximately 84 per cent by weight of carbon and approximately 14 per cent by weight of hydrogen.

These substances may be further modified by admixture with each other or with other compatible fats or oils for the purpose of modifying their physical nature. An example of such admixtures is the addition of coconut oil to a hard wax to soften it.

As will be expected, the various substances contemplated as dispersion media 'will vary among themselves in the efficiency with which they achieve the objects of this invention. This efficiency can be further modified by varying (1) the ratio of the weight of the medium to the weight of the water-soluble hormone material dispersed therein and (2) the size and shape of the resultant pelletr For most substances, we have found it desirable to use equal weights of wax and water-soluble hormone material. However, in the case of a solid fat such as oleostearin which is relatively easily penetrated by body fluids, we have found it effective and desirable to use up to nine parts of medium to one of hormone. On the other hand, in employing some of the harder waxes unmixed with a softening agent, we have found ratios of four or even five parts of hormone to one of wax to be desirable.

Using a hormone preparation of a given degree of purity (i. e., a given number of rat units of activity per milligram), fixing the dose and the ratio of wax to hormone will automatically fix the size of the pellet. The size may be varied independently of both dose and ratio, however, by the expedient of diluting the powdered hormone substance with a suitable inert, non-toxic,'watersoluble filler such as lactose or sodium chloride, and considering the total dilutedmaterial as the water-soluble hormone substance when adding yvax to the desired ratio. In general, it may be said that the greater the ratio ofwater-soluble hormone substance to waxy dispersion medium, andthe smaller the total size of the pellet, the more rapid will be absorption of-the hormone activity by the host animal; In order best to attain the objects of this invention, this absorption should be neither too rapid nor too. slow, but should, in the ordinary case, be largely accomplished within aperiod of about four to twenty days. This rate of absorption may be varied widely by the means discussed above to suit indi- 3 vidual conditions, such variations changing only the degree rather than the kind of response.

These pellets may be prepared in any convenient manner with the limitation that heat must be cautiously applied since the hormone is readily denatured and rendered inactive by excessive heating. In most cases, we have found it desirable to mix the hormone with the waxy medium while the latter is held in the molten state at the lowest possible temperature. When the resultant mass is cool, it is extruded through a die under pressure, forming a rod which can be cut into lengths of appropriate weight to contain the desired dosage. Cylindrical pellets of approximately 1.5 mm. in diameter have been found convenient to implant and to give a reasonable rate of absorption.

Example 1 The following is a specific example of how this invention may be carried out on an experimental animal. A sample of desiccated anterior pituitary gonadotropic hormone of known potency is mixed with sufiicient lactose so that the mixture has one rat unit in five milligrams of mixture, and a convenient weight of this mixture is thoroughly incorporated into an equal weight of one of the media specified in the table, warming the mass just sufficiently to permit thorough mixing. The mass is cooled, and extruded through a 1.55 millimeter opening to form a rod. This rod is out into lengths weighing 40 milligrams each, thus producing a convenient form of pellet for use in-this invention. One such pellet is implanted under the skin of each of a group of 24-day-old white rats. Ninety-six hours later, the animals are autopsied and ovarian and uterine weights deter mined. Another group of similar rats is given the same total dose of hormone dividedinto six injections of aqueous solution during three days, and autopsied 96 hours after the first injection (standard assay technique), while a third group is kept as'a control group. The table shows the effect of hormone dispersed in pellets of various media, as compared with control and standard assay groups. The figures represent the average weight in milligrams of the organ.

Per cent wax in ovarian uterine Wax v Pellet Weight Weight Mgs. Mgs. Noneleontrol, no hormone) 12.7 16.0 None (6 injections of aqueous solution in 3 days) 18.8 51. 7 Beeswax 50 41.0 112. 5

D- 90 18.0 102 Beeswax 00%, coconut oil 10%. 50 52. 2 58. Beeswax 75%, coconut oil 25% 50 62. 7 92. 8 Beeswax 90%, lecithin 10% 50 19. 7 85. Beeswax 50%, cholesterol 50% 50 37. 5 55. Oleostearin 90 38. 0 76. Paraflin 50 15. 0 72. BZ Wax A" approximately 84% by weight of carbon and 14% by Gweight of hydgogentufi f 50 32.0 48. 4

lycery monos eara e, 0 Propylene glycol monostearate, 10%. 50 0 5 Stearic acid 50 21.0 42.0

In the above examples, coconut oil is used merely as a softening agent, and may be replaced for this purpose by any bland vegetable oil.

It is clear from this example that when the hormone is administered according to this invention, ovarian and uterine weight increases are obtained which are far greater than those greater than those produced by the standard assaytechniques. Efiects of this magnitude are the more remarkable and surprising in the light of the fact that we have been unable to equal them with a many-fold increase of dose when administered by the standard assay method.

Example 2 The above demonstrations of unusual efficacy have been based on work done on experimental animals, since in that way a quantitative, objective measurement of results can be had to a degree that is impossible 'in clinical trial upon human subjects. We have nonetheless caused these pellets to be implanted in human beings. For this purpose pellets of the following different compositions have been used with equal satisfaction: (1) pellets containing 33% of beeswax and 67% of hormone substance, carrying 500 rat units of activity in a 50 milligram pellet; (2) pellets containing 50% of beeswax, of hormone substance and 20% of lactose, .carrying 200 rat units of activity in a 50 milligram pellet; (3) pelletscontaining 50% of Flexowax C (having the composition of approximately 85 by weight of carbon and 14 /2% by weight of hydrogen), and 50% of hormone substance, carrying 500 rat units of activity in a 40 milligram pellet; (4) pellets containing of BZ Wax A (having the composition of approximately 84% by weight of carbon and14% by weight of hydrogen) and 50% of'hormone substance, carrying 500 rat units of activity in a 40 milligram pellet; (5) pellets containing BZ Wax-A (having: the composition of approximately 84% by weight of carbon and 14% by weight of hydrogen) and 40% hormone substance, carrying 500 rat units of activity in a 40 milligram pellet. These pellets were prepared as described above but using proper precautions to keep the preparation sterile. The finished pellets were ap-' proximately 1.5 millimeters in diameter and 16 millimeters long, and were. preserved in sealed ampoules under 80%, alcohol. They were placed under. the skin: of either the abdomen or the side of the thigh. To accomplish the implantation,,it was frequently found convenient to insert a large-size hypodermic needle in the de-: sired site and pass the cylindrical pellet through to the end of the needle.

When thus used in a series of over thirty nonovulating and non-menstruating women, menstruation ensued after one (or occasionally two) implanation of two pellets (1000 rat units) in about 901per cent of the, cases and continued more: or less regularly for periods of many months. 1

In another series of. over thirty cases, these pellets have been. used to control functional uterine bleeding. Following a single implanation of one 500 rat unit pellet, successful responses were obtained in about per cent of the cases, successful cases being considered those in which both excessive and too frequent bleeding were inhibited and normal menses reported for at least several months following implanation of the hormone. In neither series were any deleterious effects obserbed during the treatment. We are aware that it is not new to attempt to delay the absorption of a medicament to produce beneficial results. Such delay has been accomplished by others by rendering the medicament insoluble and thus poorly absorbable; by injecting the medicament in oily solutions and suspensions; by adding to the medicament an irritant of some sort in order to cause the surrounding tissue to "wall oil the side of the injection. However, it will be obvious from a consideration of the preceding discussion that none of these methods can be effective in the present instance, as we havefurther shown experimentally. We are further aware that pellets of some of the pure steroid hormones have been successfully used, as reported by a number of investigators in recent years. In our early work we attempted to adapt such technique to the administration of the gonadotropic anterior pituitary hormone by implanting into rats pellets of this hormone, mixed only with lactose to provide sufficient bulk to handle, and found it to be completely without success, since no efiect at all was observed even though the dose given was up to one hundred times that used in the pellets of Example 1. It thus appears that the use of a wax as an excipient is necessary for the successful administration of the gonadotropic pituitary hormones in solid form.

We are further aware that pellets impreg nated with beeswax have been used to administer posterior pituitary hormones (Green and J anuary, Proceedings of the Society for Experimental Biology and Medicine, vol. 44, page 217 (1940)). Such pellets were shown by those investigators to be ineffective and undesirable as means of administration of posterior pituitary hormone, for they report, "The reaction which developed at the site of implantation was so severe that the pellets had to be removed before complete absorption occurred, and later state that this method is not applicable for treatment in man.

We have further attempted to extend the principle of this invention to the administration of the gonadotropic hormone derived from pregnant mare serum or human pregnancy urine. While not totally ineffective when applied to these hormones, we have found that the administration of wax pellets thereof presents none of the unusual advantages accruing from the use of wax pellets of the gonadotropic anterior pituitary hormones. It thus appears that the use of a wax dispersion of the gonadotropic anterior pituitary hormone, as described and claimed herein, presents a unique advantage when applied to this hormone only.

The gonadotropic fraction of the hormones of the anterior pituitary gland is known to contain more than one active principle, among which the so-called luteinizing fraction and the folliclestimulating fraction are best recognized. The presence of these two principles is demonstrated by the increases in ovarian and uterine weight, respectively, and the table in Example 1 clearly shows that the response of each is beneficially 8 afiected by administration according to the teachings of this invention.

While we have found that the cylindrical pellets referred to above are a convenient form to be used in this invention, with respect both to preparation and administration, such shape is not an essential feature of the invention. The pellets may be of any convenient shape, as, for instance, in the form of disks or ribbons or spheres, without departing from the essential features of this invention. The dose per pellet may be varied by changing either the weight of the pellet or the concentration of hormone in the pellet, or both, as illustrated in the examples. Further, while we have found the ratio of equal parts of watersoluble material and of non-absonbable medium to be convenient, and have done much of our work with pellets of such composition, we have also varied that ratio widely (as from about 10 per cent to about per cent water-soluble material), and have still been able to achieve the objects of this invention. Further, th value of this invention is not dependent upon the purity of the gonadotropic pituitary hormone employed, since it may be equally well applied to comparatively crude or to highly refined hormone preparations. The nature of the response produced may, of course, be modified by the presence of such other impurities as may not have been removed in a crude preparation. It will be understood that this invention is not limited in scope by the examples quoted herein, but is to be construed as including such equivalents as may be obvious to one skilled in the art within the scope of the appended claims.

We claim:

1. A pellet for the administration of the gonadotropic fraction of the hormones of the anterior pituitary gland by parenteral implantation, which pellet consists of a dispersion of the desiccated hormone substance in a waxy medium.

2. A pellet as in claim 1 in which the waxy medium is a naturally occurring substance.

3. A pellet as in claim 1 in which the medium is beeswax.

4. A pellet as in claim 1 in which the medium is a synthetic wax.

5. A pellet as in claim 1 in which the medium is a synthetic hydrocarbon wax having substantially the composition of /2 per cent by weight of carbon and 14 /2 per cent by weight of hydrogen.

6. A pellet as in claim 1 in which the medium is a synthetic hydrocarbon wax having substantially the composition of 84 per cent by weight of carbon and 14 per cent by weight of hydrogen.

FRANCIS J. SAUNDERS. ALBERT L. RAYMOND. 

